Colorimetric urea determination in presence of long hydrocarbon chain amidobetaine

Abstract

A colorimetric urea determination method, reagent, and reagent kit useful in end point and kinetic urea determination. Urea in a liquid sample reacts with o-phthalaldehyde and chromotropic acid or one of its salts in the presence of a linear long hydrocarbon chain amidobetaine to produce an intensely colored reaction product, the concentration of which is linearly related to the urea concentration in the sample.

We claim: 
     
       1. A method of demonstrating the presence of urea in a liquid sample, said method comprising the step of mixing said sample with a reagent comprising o-phthalaldehyde, a chromogenic compound selected from the group consisting of chromotropic acid and its salts, and a long hydrocarbon chain amidobetaine to produce a colored reaction product. 
     
     
       2. The method of claim 1 wherein said betaine is a sulfo- or carboxy-betaine. 
     
     
       3. The method of claim 2 wherein said betaine has either of the following structures: ##STR5## where R is a straight chain coconut oil residue having between about 10 and 14 carbon atoms in said chain. 
     
     
       4. A method of demonstrating the presence of urea in a sample of a human body fluid comprising the step of mixing said sample with o-phthalaldehyde and a chromogenic compound selected from the group consisting of chromotropic acid and its salts, under slightly acidic conditions in the presence of an amidobetaine having either of the following structures: ##STR6## where R is a straight chain coconut oil residue having between about 10 and 14 carbon atoms in said chain, to produce a colored reaction product.   
     
     
       5. A method of determining the urea concentration is a liquid sample comprising the steps of: (a) adding o-phthalaldehyde, a chromogenic compound selected from the group consisting of chromotropic acid and its salts, and a long hydrocarbon chain amidobetaine to said liquid sample;   (b) maintaining said sample at a temperature at which said chromogenic compound, said o-phthalaldehyde and said urea in said sample react to produce a colored reaction product;   (c) obtaining a colorimetric absorbance reading for said sample; and   (d) comparing said absorbance reading with calibration means.   
     
     
       6. The method of claim 5 wherein said chromogenic compound comprises chromotropic acid. 
     
     
       7. The method of claim 5 wherein said chromogenic compound comprises the disodium salt of chromotropic acid. 
     
     
       8. The method of claim 5 wherein said amidobetaine has either of the following structures: ##STR7## where R is a straight chain coconut oil residue having between about 10 and 14 carbon atoms in said chain. 
     
     
       9. In a method of determining the urea concentration in a liquid sample comprising the steps of mixing said sample with o-phthalaldehyde and a chromogenic compound selected from the group consisting of chromotropic acid and its salts under slightly acidic conditions to form a colored reaction product, obtaining a colorimetric absorbance reading of said sample and comparing said absorbance reading with calibration means, the improvement wherein said colored reaction product is formed in the presence of a long hydrocarbon chain amidobetaine. 
     
     
       10. The method of claim 9 wherein said amidobetaine has either of the following structures: ##STR8## where R is a straight chain coconut oil residue having between about 10 and 14 carbon atoms in said chain. 
     
     
       11. A method of determining the urea concentration of a liquid sample, said method comprising the steps of: (a) adding said sample to a sample container;   (b) adding equal amounts of a reagent comprising a solution of o-phthalaldehyde, a chromogenic compound selected from the group consisting of chromotropic acid and its salts, and a long hydrocarbon chain amidobetaine to said sample container and a blank container;   (c) maintaining the contents of each said container at a temperature at which said o-phthalaldehyde and said urea in said sample react to produce a colored reaction product;   (d) obtaining first colorimetric absorbance readings for each of said blank and sample containers at the end of a first time period;   (e) obtaining second colorimetric absorbance readings for each of said blank and sample containers at the end of a second time period;   (f) computing the difference between said first and second absorbance readings for each of said sample and blank containers; and,   (g) comparing said differences with calibration means.   
     
     
       12. The method of claim 11 wherein said amidobetaine is selected from the group consisting of betaines having the following structures: ##STR9## where R is a straight chain coconut oil residue having between about 10 and 14 carbon atoms in said chain. 
     
     
       13. A reagent for colorimetric urea determination consisting essentially of a solution of (a) o-phthalaldehyde, (b) a chromogenic compound selected from the group consisting of chromotropic acid and its salts, and (c) a long hydrocarbon chain amidobetaine. 
     
     
       14. A reagent for colorimetric urea determination consisting essentially of an acidic solution of (a) o-phthalaldehyde, (b) a chromogenic compound selected from the group consisting of chromotropic acid and its salts, and (c) a long hydrocarbon chain amidobetaine selected from the group consisting of compounds of the formula ##STR10## where R is a straight chain coconut oil residue having between about 10 and 14 carbon atoms in said chain. 
     
     
       15. A reagent kit for colorimetric determination of urea, said reagent kit consisting of a package containing a first container containing a first solution comprising a colorimetric amount of a chromogen selected from the group consisting of chromotropic acid and its salts, and a second container containing a second solution comprising a colorimetric amount of o-phthalaldehyde, one of said first and second solutions further comprising a long hydrocarbon chain amidobetaine. 
     
     
       16. The reagent kit of claim 15 wherein said amidobetaine is selected from the group consisting of compounds of the formula ##STR11## where R is a straight chain coconut oil residue having between about 10 and 14 carbon atoms in said chain.